Citation for published version:Parveen, I, Wilson, T, Donnison, I, Cookson, AR, Hauck, B & Threadgill, M 2013, 'Potential sources of highvalue chemicals from leaves, stems and flowers of Miscanthus sinensis 'Goliath' and Miscanthus sacchariflorus',Phytochemistry, vol. 92, pp. 160-167. https://doi.org/10.1016/j.phytochem.2013.04.004

DOI:10.1016/j.phytochem.2013.04.004

Publication date:2013

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1

Potential sources of high value chemicals from leaves, stems and flowers of

Miscanthus sinensis ‘Goliath’ and Miscanthus sacchariflorus

Ifat Parveena,, Thomas Wilsona, Iain S. Donnisona, Alan R. Cooksona, Barbara Haucka,

and Michael D. Threadgillb

aInstitute of Biological, Environmental and Rural Sciences, Aberystwyth University,

Gogerddan, Aberystwyth SY23 3EB, UK

bMedicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath,

Claverton Down, Bath BA2 7AY, UK

Abstract

Society demands chemicals from sustainable sources. Identification of commercially

important chemicals in crops increases value in biorefineries and reduces reliance on

petrochemicals. Miscanthus sinensis and Miscanthus sacchariflorus are high-yielding

distinct plants, which are sources of high-value chemicals and bioethanol through

fermentation. Cinnamates in leaves, stems and flowers were analysed by LC-ESI-MSn.†

Corresponding author: Tel: +44 (0)1970 823216; Fax: +44 (0)1970 828357; E-mail: ifp@aber.ac.uk †Abbreviations: ADME = absorption, disposition, metabolism, excretion; CADPE = 2-(3,4-dihydroxyphenyl)ethyl caffeate; CAPE = 2-phenylethyl caffeate; 2-CaffHydCitA = 2-caffeoylhydroxycitric acid; 3-p-CoQA = 3-para-coumaroylquinic acid; 4-p-CoQA = 4-para-coumaroylquinic acid; 5-p-CoQA = 5-para-coumaroylquinic acid; CaffQA = caffeoylquinic acid; 1-CaffQA = 1-caffeoylquinic acid; 3-CaffQA = 3-caffeoylquinic acid; 4-CaffQA = 4-caffeoylquinic acid; 5-CaffQA = 5-caffeoylquinic acid; CaffSA = caffeoylshikimic acid; 3-CaffSA = 3-caffeoylshikimic acid; 4-CaffSA = 4-caffeoylshikimic acid; 5-CaffSA = 5-caffeoylshikimic acid; 2-CaffTA = 2-caffeoylthreonic acid; 2,3-diHydBA = 2,3-dihydroxybenzoic acid; 2,5- diHydBA = 2,5-dihydroxybenzoic acid; 3,4- diHydBA = 3,4-dihydroxybenzoic acid; 2,3- diHydBA = 2,3-dihydroxybenzoic acid hexoside; 3,4-diHydBAHex = 3,4-dihydroxybenzoic acid hexoside; 1,4-diCaffQA = 1,4-dicaffeoylquinic acid; 1,5-diCaffQA = 1,5-dicaffeoylquinic acid; 3,4-diCaffQA = 3,4-dicaffeoylquinic acid; 3,5-diCaffQA = 3,5-dicaffeoylquinic acid; FA = ferulic acid; 2-FHydCitA = 2-feruloyl hydroxycitric acid; 3-FQA = 3-feruloylquinic acid; 4-FQA = 4-feruloylquinic acid; 5-FQA = 5-feruloylquinic acid; 2-FTA = 2-O-feruloylthreonic acid; FW = fresh weight; 2-HydBA = 2-hydroxybenzoic acid; 3-HydBA = 3-hydroxybenzoic acid; 4-HydBA = 4-hydroxybenzoic acid; HydBAHex = hydroxybenzoic acid hexoside; HIV-1 = human immunodeficiency virus-1; HPLC = high-performance liquid chromatography; LC-ESI-

2

Free phenols were extracted and separated chromatographically. More than twenty

hydroxycinnamates were identified by UV and LC-ESI-MSn. Several cinnamate

hexosides were detected in the M. sinensis flower and in M. sacchariflorus (leaf and

stem). Hydroxybenzoic acids and their hexosides were observed in leaf and stem of M.

sacchariflorus. Higher concentrations of 3-feruloylquinic acid were observed in M.

sacchariflorus stem, suggesting a role in cell-wall biosynthesis. This technique can be

used to screen plants in a mapping family to identify genotypes / species with high

concentrations of phenols. Plants with low concentrations of antimicrobial phenols may

be good feedstocks for fermentation.

Highlights

Miscanthus sinensis & sacchariflorus: sources of biofuel & platform chemicals

LC-ESI-MSn identified >20 hydroxycinnamates; flowers contained cinnamate

hexosides

Hydroxybenzoic acids and hexosides observed in leaf & stem of M. sacchariflorus

More 3-FQA in M. sacchariflorus stem suggests a role in cell-wall biosynthesis

Plants with low antimicrobial phenols may be good feedstocks for fermentation

Keywords: Miscanthus sinensis; Miscanthus sacchariflorus; hydroxybenzoic acids;

hydroxycinnamate esters; high-value chemicals.

MS = liquid chromatography-electrospray ionisation mass spectrometry; p-CoA = para-coumaric acid; PDA = photodiode array; SA = syringic acid; UV = ultra-violet.

3

1. Introduction

Concerns about global warming and depletion of fossil fuels have stimulated interest in

development of cleaner technologies that use sustainable and carbon-neutral feedstocks

(Alonso et al., 2010; Sims et al., 2006). Sustainable feedstocks will be provided, at least

in part, by the cultivation of non-food energy crops (Lewandowski et al., 2000; Sims et

al., 2006). Miscanthus is a genus consisting of ca. fifteen species of perennial grasses

that are native to tropical and subtropical regions of Africa, South Asia and temperate

zones of Asia (Villaverde et al., 2010a, 2010b). Miscanthus giganteus (M.

giganteus), a perennial rhizomatous grass with C4-photosynthesis, can grow to heights

of more than 3.5 m in a single growth season (Villaverde et al., 2010a, 2010b). This

hybrid is a sterile triploid formed by a cross between Miscanthus sacchariflorus (M.

sacchariflorus) and Miscanthus sinensis (M. sinensis). In this paper, we evaluate and

compare the phenolic composition of these two claimed parent species of this hybrid.

There is much interest in using Miscanthus as an alternative to food crops and

petroleum-based feedstocks to make a variety of bulk, intermediate and speciality

chemicals, in addition to providing fuel. Miscanthus plants can grow under a range of

climatic and environmental conditions (Lewandowski et al., 2000). After senescence,

the plant can be burned for heat and electricity or fermented to produce biofuel. The

chemical composition can affect the efficiency of conversion of biomass to energy and

to chemical products (Klinke et al., 2004). Soluble phenols inhibit the bioconversion of

sugars to ethanol, as many are toxic to the fermenting microorganisms (Klinke et al.,

2004); thus it is important to profile the content of these unwanted components.

4

Plants produce a wide range of monophenolic and polyphenolic compounds with roles

in strengthening cell walls, protection against UV radiation, tolerance to stress and

resistance to pathogens. Hydroxycinnamic acids are the most widely distributed group

of secondary compounds and are present as free, conjugated-soluble and bound-

insoluble forms. They acids are often found as glycosides, sugar esters and amides.

Caffeate esters predominate in grass, the most abundant being 5-O-caffeoylquinic acid.

Hydroxycinnamic acids (specifically ferulic and p-coumaric acids) are covalently linked

into the cell wall of grass species, where they cross link hemicellulose and lignin.

The potential benefits of these compounds are now appreciated by food and cosmetic

industries (Dimitrios et al., 2006; Naczk and Shahidi, 2006, 2004; Ou et al., 2004;

Padilla et al., 2005). Pharmaceutical companies continue to use natural products as

sources of leads for the development of drugs (Harvey, 2008; Ou et al., 2004) and

hydroxycinnamic acid conjugates are considered important drug leads (Touaibia et al.,

2011). Many are reducing agents, hydrogen-donating antioxidants and quenchers of

singlet oxygen (Parveen et al., 2011); thus diverse phenolic derivatives have interesting

biological activities (Touaibia et al., 2011).

We recently reported the use of LC-ESI-MSn to profile rapidly more than twenty

phenols in the leaf and stem tissues of the hybrid M. giganteus, including several

novel mandelonitriles and mandelamides (Parveen et al., 2011). Total phenol

concentration (hydroxycinnamates) in the leaf tissue was 0.04% FW and in the stem

tissue 0.02% FW. The analysis was carried out on green material when the plant was at

its highest biomass. Here we use LC-ESI-MSn for qualitative and quantitative profiling

of hydroxycinnamates in leaf, stem and flower extracts of the parental lines M. sinensis

5

and M. sacchariflorus at the same high biomass stage of growth as was M. giganteus,

to draw comparisons and conclusions about the relative utilities of these plants.

Knowledge of the content and composition of the soluble phenols chemistry of these

plants would help to determine the potential for exploiting this plant.

2. Results and discussion

2.1. Extraction of phenols

Soluble phenols were obtained from the methanol extract of fresh leaves, stems and

flowers of M. sinensis and leaves and stems of M. sacchariflorus. More than twenty

phenols (including hydroxycinnamic acid conjugates) were identified with UV

absorptions typically in the region (240-340 nm) (Tables 2 and 3 (Supplementary

Information) and Figures 1 and 2). The UV absorbance maxima could not be recorded

for some cinnamic acid conjugates, either owing to low abundance or because they were

masked by co-eluting peaks. The identification method used UV absorption coupled

with the fragmentation pattern observed in tandem mass spectra using LC-ESI-MSn.

Where possible, the mass spectra and retention times of the phenols were compared

with those of standards, including caffeic acid, o-coumaric acid, p-coumaric acid,

cinnamic acid, ferulic acid, syringic acid, 5-caffeoylquinic acid (5-CaffQA, also known

as chlorogenic acid), sinapic acid, 2-, 3-, and 4-hydroxybenzoic acid (2-HydBA, 3-

HydBA and 4- HydBA), 2,3, 2,5- and 3,4-dihydroxybenzoic acid (2,3-diHydBA, 2,5-

diHydBA and 3,4-diHydBA), 2-hydroxy-3-methoxybenzoic acid, 2-hydroxy-4-

methoxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid (vanillic acid). In the

absence of commercial standards, caffeoyl-, p-coumaroyl- and feruloyl- conjugates

6

were assigned primarily by their parent ion; their UV spectrum and elution order and

assignments were supported by comparison of their mass spectrometric fragmentation

data (MS2 and MS3) to those previously reported (Clifford et al., 2005; Jaiswal et al.,

2010; Parveen et al., 2008;). In the absence of standards, all other identifications are

considered provisional.

2.2. Characterisation of caffeoyl derivatives

This is the first study on the HPLC profiles of phenols in M. sinensis and M.

sacchariflorus tissues. In all tissues, the most abundant compound was 5-O-

caffeoylquinic acid (5-CaffQA). Particularly high concentrations of 3-O-caffeoylquinic

acid (3-CaffQA) were detected in M. sacchariflorus tissues (Table 1 and Tables 2 and 3

(Supplementary Information)). The pattern of fragmentation and retention time for 5-

CaffQA were consistent with those of a commercial standard. Diagnostic fragmentation

ions of caffeoylquinic acids in negative-ion mode ESI-MSn involved one of two

pathways (i) loss of the acyl group with cleavage of the carbonyl-oxygen bond, (ii) -

elimination of a carboxylic acid (Parveen et al., 2011). Geometrical isomers were

evident; for example, cis / trans isomers of caffeic acid were found in all extracts, based

on photoirradiation experiments resulting in photo-isomerisation (Parveen et al., 2011).

As observed previously in the stem extract of M. sinensis, a small but distinct peak

showed properties very similar to that of 5-CaffQA in MS/MS negative-ion mode

spectra. This was evidently a CaffQA -stereoisomer but 1-CaffQA was discounted, as

this peak did not co-elute with an authentic sample of 1-CaffQA from acid-catalysed

hydrolysis of cynarin (1,3-dicaffeoylquinic acid) (Parveen et al., 2011).

7

In M. sinensis leaf and M. sacchariflorus leaf and stem tissues, minor compounds

giving peaks at m/z 335 [M - H]- and ions at m/z 317, 291, 179, 161, 135 were

consistent with O-caffeoylshikimic acids (CaffSA) (Jaiswal et al., 2010). As for quinic

acid, shikimic acid can form esters with cinnamic acids. Jaiswal et al. (2010) reported

the synthesis and fragmentation pathways of 3-, 4-, and 5-CaffSA in negative ion mode.

CaffSAs have been widely reported in plants (Fang et. al., 2002; Jaiswal et al., 2010);

however, they were minor components in the Miscanthus tissue extracts. Three minor

components with pseudomolecular ion 515 (M - H)- were detected in the leaf of M.

sacchariflorus and were identified by their fragmentations patterns as 1,4-diCaffQA,

3,4-diCaffQA and 3,5-diCaffQA (Clifford et al., 2007). Dicaffeoylquinic acids have

previously been reported in M. giganteus and in herbal chrysanthemum (Clifford et

al., 2007; Parveen et al., 2011). Three further compounds in M. sinensis flowers and leaf

and stem of M. sacchariflorus with pseudomolecular ion 515 [M - H]- were clearly

hydroxycinnamates but not dicaffeoylquinic acids. The MS2 experiment yielded ions

m/z 353, 341, 323, 191 and 179. These compounds were tentatively identified as

caffeoylquinic acid hexosides where the hexose is linked to a hydroxy on a caffeoyl

moiety as a glycoside; the linkage hexose-caffeoyl-quinic acid is demonstrated by the

observation of fragment ions corresponding to CaffQAs. These compounds were

previously reported in the hybrid M. giganteus (Parveen et al., 2011). In M.

sacchariflorus, compounds showing pseudomolecular ions and fragmentation patterns

in negative-ion mode m/z 297 [M - H]- and 369 [M - H]- corresponded to 2-O-

caffeoylthreonic acid (2-CaffTA) and 2-O- acid caffeoyloxycitric (2-CaffHydCitA),

respectively. These compounds have previously been reported in Dactylis glomerata

and in Cornus controversa (Lee et al., 2000; Parveen et al., 2011). Other caffeoyl-

8

derivatives were observed in M. sacchariflorus but these could not be identified.

Examples of structures are shown in Figure 3.

2.3. Characterisation of p-coumaroyl derivatives

The patterns of fragmentation observed at MS2 and MS3 enabled the identification of

three trans- isomers of O-p-coumaroylquinic acid (3-p-CoQA, 4-p-CoQA and 5-p-

CoQA) in M. sinensis and M. sacchariflorus tissue extracts. The 5-acylated isomer was

characterised by a MS2 base ion m/z 191; 3-p-CoQA yielded MS2 base peak m/z 163

[coumarate]- and 4-p-CoQA was distinguished by -elimination of coumaric acid to

give MS2 base ion m/z 173. Several isomers with pseudomolecular ions 325 [M - H]-,

MS2 base ion m/z 179 and secondary ions m/z 135, 281, 251 and 221 were identified as

a p-coumaric acid hexoside in the flowers of M. sinensis and leaf and stem of M.

sacchariflorus (Tables 2 and 3 (Supplementary Information)). Losses of 30, 60 and 90

are typical losses of CHOH units from sugars. Furthermore, in the flower extract, a

minor distinct peak at tR 9.3 min also gave a pseudomolecular ion m/z 341 [M - H]- in

the MS2 spectrum. The UV absorption spectrum was not characteristic of O-

acylquinates but rather of a cinnamic acid dimer (Pati et al., 2006). Thus we tentatively

assign this compound as a caffeic acid dimer (Table 2). In both leaf and stem tissue of

M. sinensis, a peak with a pseudomolecular ion m/z 389 [M - H]- was detected in

negative-ion mode MS. An MS2 experiment yielded a major ion at m/z 371 and

secondary ions at m/z 327, 297, 163 and 119. We can only speculate that this compound

is a p-coumaroyl linked via an ester to a sugar unit comprising at least one carboxylic

acid.

9

2.4. Characterisation of feruloyl derivatives

In M. sinensis and in M. sacchariflorus tissues, three trans-isomers of O-feruloylquinic

acids (3-FQA, 4-FQA and 5-FQA) were readily identified by their pseudomolecular ion

m/z 367 [M - H]-; 3-FQA was distinguished by its MS2 base ion m/z 193 [ferulate]-,

while 4-FQA was discriminated by the abundant peak m/z 173 [M – ferulic acid]- (from

-elimination) and the 5-isomer was characterised by the dominant fragment m/z 191

[quinate]- (Tables 2 and 3 (Supplementary Information)). Two geometrical isomers of 2-

O-feruloyl-hydroxycitric acid (2-FHydCitA) were identified in the leaf and stem of M.

sacchariflorus by their pseudomolecular ions [383]- and fragmentation ions 207, 193

and 189. This compound has been previously reported in Dactylis glomerata as a

component of Zea mays (Ozawa et al., 1977; Parveen et al., 2008). In both plants,

compounds with pseudomolecular ions 355 [M - H]-, MS2 ion m/z 193 and losses of 30,

60 and 90 corresponding to CHOH units were tentatively identified as ferulic acid

hexosides.

2.5. Characterisation of other phenolic derivatives

Other minor components identified in M. sacchariflorus extract were a syringic acid

hexoside identified by its pseudomolecular ion [359]- and MS2 ions 197, 182 and 153

corresponding to the loss of a proton, a methyl group and a carboxylic acid (Tables 2

and 3 (Supplementary Information)). These compounds have previously been reported

as constituents in dried plums (Fang et al., 2002). These compounds were only

identified in M. sacchariflorus extracts. Standards were used for the identification of

several hydroxybenzoic acids (HydBAs) and identified on the basis of MS data and LC

retention times under conditions described above. These were 4-HydBA, 2,3-diHydBA,

10

3,4-diHydBA, vanillic acid and their sugar glycosides. These were detected in relatively

high concentrations in leaves and stems of M. sacchariflorus.

2.6. Quantification and comparison of abundances

The abundances of selected compounds in the various extracts were by measurement of

peak area in the HPLC chromatogram and reference to the extinction coefficient of

chlorogenic acid. Data are presented in Table 1. Total hydroxycinnamates in M. sinensis

were leaf = 1.2% FW; stem = 0.4% FW; flower = 0.7% FW and in M. sacchariflorus

were leaf = 1.0% FW and stem = 1.1% FW.

Caffeoylquinic acids were the most abundant phenolic metabolites in all tissues

examined; this was also true of the hybrid M. × giganteus (Parveen et al., 2011).

However, there were marked differences in abundance of individual regioisomers

between the species and between different tissues. For example, 3-CaffQA 1 was

present to only 0.18-0.68 mol g-1 in M. sinensis, whereas it was more than an order of

magnitude more abundant in M. sacchariflorus (7.0 mol g-1 in leaf and 4.5 mol g-1 in

stem). It was also of low abundance in M. × giganteus (Parveen et al., 2011). By

contrast, 4-CaffQA 14 was much more abundant in M. sinensis (12 mol g-1 in leaf, 3.0

mol g-1 in stem and 5.8 mol g-1 in flower) that it was in M. sacchariflorus (2.6 mol

g-1 in leaf and not quantifiable in stem). 5-CaffQA 9 was present in high concentrations

(3.3-13 mol g-1) in all tissues of M. sinensis and M. sacchariflorus examined. The total

CaffQAs for the putative parental species were 28 mol g-1 (leaf) and 4.3 mol g-1

(stem) for M. sinensis and 23 mol g-1 (leaf) and 17 mol g-1 (stem) for M.

11

sacchariflorus, values which are much greater than those for the progeny M. ×

giganteus (1.0 mol g-1 and 0.1, respectively) (Parveen et al., 2011).

Coumaroylquinic acids 4,13,15,19,25 were present at much lower levels than were the

CaffQAs in all tissues of M. sinensis and M. sacchariflorus; this is parallelled in the

progeny M. × giganteus (Parveen et al., 2011). The picture is rather different for the

feruloylquinic acids. Most of these are present in relatively low abundance in M.

sinensis leaf (0.8-0.9 mol g-1), M. sinensis stem (0.5-1.1 mol g-1) and M.

sacchariflorus leaf (0.2-1.3 mol g-1) but 3-FQA 11 was found at the remarkably high

abundance of 7.7 mol g-1 in the stem of M. sacchariflorus. The total FQAs present in

M. × giganteus stem were 3 nmol g-1. Ferulic acid is known to be involved in

biosynthesis of cell walls (Ralph et al., 1994) and these results may suggest that M.

sacchariflorus is a more woody plant than are M. sinensis and M. sacchariflorus.

Miscanthus species generally have a high lignin content and have been described as

being closer in structure to a woody material than to a grass (Villaverde et al., 2010b,

2010c). Hydroxycinnamic acids, specifically ferulic acid and para-coumaric acid, are

covalently linked into the cell wall of grass species, where they cross link hemicellulose

and lignin by ester and ether bonds. It has been reported that their concentration in the

cell wall is inversely correlated with recalcitrance of biomass to hydrolysis /

saccharification (Akin, 2007). Microscopy of the stem tissue from M. sinensis, M.

sacchariflorus and M. × giganteus correlated with the much higher amount of 3-FQA,

in that the M. sacchariflorus stem had a considerably more woody structure. Figure 4

shows thin sections of the stems of M. sinensis, M. sacchariflorus and M. giganteus

stained with phloroglucinol to show cinnamic lignin. In M. sinensis, the lignin staining

tends to be associated only with vascular bundles, whereas in M. giganteus it is

12

somewhat more extensive, with some in the parenchyma. In M. sacchariflorus, almost

all cells contain lignified precursors.

Caffeoylshikimic acids 20,21,24,26,68,75,76 were evident in M. sinensis leaf and M.

sacchariflorus leaf and stem at low abundances; other acylshikimic acids were not

detected. Small amounts of a wide range of hexosides were also observed.

Previously, we have shown that M. giganteus stem extract contains a significant

amount (0.3 mol g-1) of a mandelonitrile-CaffQA (Parveen et al., 2011). These were

not detected in either of the two putative parent plants (M. sinensis and M.

sacchariflorus). By contrast, we were unable to detect hydroxybenzoic acid derivatives

in M. giganteus but a number of derivatives were present in the two parental species

in modest abundances (up to 1.3 mol g-1 for 40 in M. sacchariflorus stem).

Hydroxybenzaldehyde was observed as a major component in the stem extract of M.

giganteus but was absent from the parental species. Syringic acid hexoside was also

observed in M. sacchariflorus extracts. Could some of these compounds be markers for

these plant species?

2.7. Applications

Phenols synthesised by plants are the most widespread antioxidants and 5-CaffQA is

reported to accumulate to high concentrations in grasses such as Miscanthus (Parveen et

al. 2008, 2011). Desirable properties of 5-CaffQA in the pharmaceutical and food

industries include antioxidant, anti-carcinogenic, anti-inflammatory and cholesterol-

lowering activities (Parveen et al., 2011). In addition, 5-CaffQA has been reported to

protect against degenerative age-related diseases in animals when offered diets rich in

13

this compound (Niggeweg et al., 2004). Tomatoes have also been engineered with

increased amounts of 5-CaffQA, with the potential to improve human health (Niggeweg

et al., 2004). Caffeoylquinic acids are abundant in a wide range of fruit and vegetables

and it has been claimed that up to 80% of cardiovascular disease, 90% of type II

diabetes and one third of all cancers could be avoided by changes in lifestyle, in

particular, changes to diet (Fraga, 2009). There is much epidemiological evidence that a

diet rich in phenols can reduce incidences of such non-communicable diseases (Crozier

et al., 2009). Caffeoylquinic acids have also been reported recently to protect against the

aggregation and neurotoxicity of the 42-residue amyloid -protein (Miyamae et al.,

2012), although some issues in ADME have yet to be resolved.

Caffeic acid has a potential role in functional foods or pharmaceuticals due to its

antioxidant, anti-inflammatory and anti-cancer properties (Cambie and Ferguson 2003;

Touaibia et al., 2011). Caffeoyl esters, such as 2-phenylethyl caffeate (CAPE) inhibit of

HIV-1 integrase (Fesen et al., 1994). By contrast, 2-(3,4-dihydroxyphenyl)ethyl caffeate

(CADPE) inhibits growth and formation of colonies by HGC27 (gastric cancer), H1299

(lung carcinoma), A549 (lung carcinoma), HCT116 (colon cancer), and U2OS

(osteosarcoma) human cancer cells through inducing senescence by increasing cellular

size and cytoplasmic granularity. CADPE appeared to suppress significantly the

expression of Twist 1, which is responsible for the up-regulation of p53, p21 and p16

proteins, in these lines (Dong et al., 2011). Both CAPE and CADPE have been isolated

from Sarcandra glabra and Teucrim pilosum and have been used as an alternative to

cytotoxic cancer therapy (Dong et al., 2011).

14

Large differences in abundances of some of the metabolites (as noted above) indicate

that these might be developed as biomarkers for identification of Miscanthus species.

The markedly different profiles of some metabolites also raise the question of whether

M. sinensis and M. sacchariflorus are indeed the parents of M. × giganteus.

3. Conclusion

Profiles are reported of more than twenty hydroxycinnamates in M. sinensis and M.

sacchariflorus, parental lines of M. giganteus. Profile differences facilitate screening

mapping populations, linking soluble chemotype populations with quality traits loci.

This enables screening of plants in a mapping family to identify genotypes / species

with high concentrations of valuable phenols. Plants with few antimicrobial phenols are

good fermentation feedstocks. Knowledge of the phenolic profile in high biomass-

yielding Miscanthus species can facilitate conversion into industrially useful products.

Moreover, quinic acid is a key precursor of the anti-influenza drug oseltamavir

(Tamiflu), suggesting Miscanthus as an alternative and reliable source. This early

comparative study provides information on the content of potentially valuable

compounds from readily renewable sources and possible biomarkers for identification.

4. Materials and methods

4.1. Plant materials

M. sinensis (cv. Goliath) and M. sacchariflorus were sown in experimental plots (20 m

20 m) in 2009 at the Institute of Biological, Environmental and Rural Sciences

(IBERS), Aberystwyth, UK. Bulk plant material was harvested (July 2009); leaves,

15

stems and flowers were separated, weighed and stored at -70°C for extraction and

analysis.

4.2 Extraction and purification of phenols

Methanolic extracts were prepared as previously reported (Parveen et al., 2008).

Samples were analysed by HPLC (Figures 1 and 2).

4.3. Analytical HPLC

The Waters HPLC system (Waters Corporation, USA) included an auto-sampler and a

996 photodiode array (PDA) detector coupled to an analytical workstation. The column

configuration consisted of a Waters C18 reversed-phase Nova-Pak cartridge (4.0 m, 8.0

mm 100 mm). The sample injection volume was 25 L.

The detection wavelength was set at 240–400 nm, the flow rate was 2.0 ml min-1, the

auto-sampler tray temperature was kept at 4ºC and the column was at room temperature.

The mobile phase comprised purified water-acetic acid (A; 95:5, v/v) and HPLC grade

methanol (B). The initial condition was A:B (95:5, v/v) and the percentage of mobile

phase B increased linearly to 100% over the 55 min run. Phenols were detected with a

PDA detector at two wavelengths (λmax 280 and 340 nm) and individual peaks were

collected and solvents were evaporated under reduced pressure in a rotary evaporator.

The software Empower (Waters Corporation, USA) was run on a Pentium III PC.

16

4.4. LC-ESI-MSn analysis

The Thermo Finnigan HPLC/MSn system (Thermo Electron Corporation, USA)

included an on-line degasser, an auto-sampler, a column temperature controller, a

photodiode array detector and a linear ion trap with ESI source, coupled to an analytical

workstation. The configuration consisted of a Waters C18 reversed-phase Nova-Pak

column (4.0 m, 3.9 mm 100 mm). The sample injection volume was typically 10 μL.

The detection wavelength was set at 240-400 nm, the flow rate was 1.0 mL min-1, with

100 μL min-1 going to the mass spectrometer. The auto-sampler tray temperature was

kept at 4ºC and the column temperature was maintained at 30ºC. The mobile phase

consisted of purified water (A) and HPLC grade methanol-formic acid (B; 1000:1). The

initial condition was A:B (95:5, v/v) and the percentage of mobile-phase B increased

linearly to 100% over the 55 min run. The mass spectra were acquired. N2 was used as

the sheath and auxiliary gas and He was used as the collision gas. For the phenolic

acids, the ionisation mode was negative and the interface and MSD parameters were as

follows: sheath gas, 30 arbitrary units; auxiliary gas, 15 units; spray voltage, 4.0 KV;

capillary temperature 320ºC; capillary voltage, 1 V; tube lens offset, 68 V.

4.5. Staining of stem sections

Free-hand sections of the stems were stained with 1% w/v phloroglucinol in aq. HCl

(6.0 M). Sections were observed at 10 magnification on a Leica DM6000B microscope

with a Hitachi HV-D20 camera.

17

Supplementary Information

Table 2. HPLC / ESI-MS / MS (negative-ion mode) characterisation of hydroxy-

cinnamates in extracts of M. sinensis leaf, stem and flower.

Table 3. HPLC / ESI-MS / MS (negative-ion mode) characterisation of hydroxy-

cinnamates in extracts of M. sacchariflorus leaf and stem.

Acknowledgements

We thank the Biotechnology and Biological Sciences Research Council for financial

support. BEACON is supported by the European Regional Development Fund through

the Welsh European Funding Office, part of the Welsh Assembly. We also thank Dr.

Ana Winters, Sue Youell and Charlotte Hayes (Aberystwyth) for helpful discussions.

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Captions for Figures

Figure 1. HPLC/UV (340 nm) chromatograms of phenolic components in extracts of

Miscanthus sinensis leaf, stem and flower. 1, 3-CaffQA (trans); 2, p-coumaric acid

hexoside (trans), 3, caffeic acid dimer (trans); 4, 3-p-CoQA (trans); 5, 4-CaffQA (cis);

6, caffeic acid hexoside (trans); 7, CaffQA stereoisomer (trans); 8, 3-FQA (cis); 9, 5-

CaffQA (trans); 10, caffeic acid hexoside (trans); 11, 3-FQA (trans); 12, ferulic acid

hexoside (trans); 13, 4-p-CoQA (cis); 14, 4-CaffQA (trans); 15, 5-p-CoQA (trans); 16,

p-coumaric acid hexoside (trans); 17, 5-CaffQA (cis); 18, 4-FQA (cis); 19, 4-p-CoQA

(trans); 20, 5-CaffSA (trans); 21, 3-CaffSA (trans); 22, 5-FQA (trans); 23, 4-FQA

(trans); 24, 5-CaffSA (cis); 25, 5-p-CoQA (cis); 26, 3-CaffSA (cis); 27, 5-FQA (cis);

28, p-coumaroyl ester.

22

Figure 2. HPLC/UV (340 nm) chromatograms of phenolic components in extracts of

Miscanthus sacchariflorus leaf and stem. 29, 4-hydroxybenzoic acid; 30, 4-

hydroxybenzoic acid hexoside; 31, 3,4-dihydroxybenzoic acid hexoside; 32, 3,4-

dihydroxybenzoic acid hexoside; 33, 2,5-dihydroxybenzoic acid; 34, feruloyl-

hydroxycitric acid; 35,-3-CaffQA (cis); 36, 3,4-dihydroxybenzoic acid hexoside; 37, -4-

hydroxybenzoic acid hexoside; 38, 3-hydroxybenzoic acid; 39, 3,4-dihydroxybenzoic

acid hexoside; 40, vanillic acid hexoside; 41, feruloyl-hydroxycitric acid; 42, 2-

hydroxybenzoic acid; 1, 3-CaffQA (trans); 33, 4-CaffQA hexoside (trans); 44, 2-

CaffHydCitA (trans), 45, 3,4-dihydroxybenzoic acid hexoside; 46, vanillic acid

hexoside; 47, 2,3-dihydroxybenzoic acid; 48, 3-CaffQA hexoside (trans); 49, quinoyl-p-

coumaric acid (trans); 50, 4-hydroxybenzoic acid hexoside; 51, caffeic acid hexoside

(trans); 52, syringic acid hexoside (trans); 53, p-coumaric acid hexoside (trans); 5, 4-

CaffQA (cis); 4, 3-p-CoQA (trans); 54, syringic acid (trans) hexoside; 55, caffeic acid

hexoside (trans); 56, syringic acid hexoside (trans); 57, vanillic acid; 58, 1,5-diCaffQA

(trans); 59, p-coumaric acid hexoside; 8, 3-FQA (cis); 60, caffeic acid hexoside; 10,

caffeic acid hexoside (trans); 61, unknown hexose / caffeic acid; 62, ferulic acid (trans),

11, 3-FQA (trans); 12, ferulic acid hexoside (trans); 63, unknown hexose / caffeic acid;

9, 5-CaffQA (trans); 64, caffeic acid hexoside; 65, 3-CaffQA hexoside; 66, isovanillic

acid; 67, caffeic acid hexoside; 14, 4-CaffQA (trans); 68, 4-CaffSA; 69, p-coumaric

acid; 70, unknown caffeoyl compound; 71, ferulic acid hexoside (trans); 72, CaffQA

stereoisomer; 73, 2-CaffTA (trans); 18, 4-FQA (cis); 19, 4-p-CoQA (trans); 17, 5-

CaffQA (cis); 15, 5-p-CoQA (trans); 74, unknown hexose / caffeic acid; 75, 4-CaffSA

(trans) (trans); 23, 4-FQA (trans); 25, 5-p-CoQA (cis); 76, CaffSA (trans); 22, 5-FQA

(trans); 77, unknown hexose / caffeic acid; 78, unknown hexose / caffeic acid; 26, 3-

23

CaffSA (cis); 27, 5-FQA (cis); 79, CaffHydCitA (trans); 80, unknown hexose / caffeic

acid; 81, p-CoSA; 82, caffeic acid hexoside (trans); 83, unknown hexose / caffeic acid;

84, unknown hexose / caffeic acid; 85, unknown hexose / caffeic acid; 86, 3,4-

diCaffQA (trans); 87, 3,5-diCaffQA (trans); 88, 1,4-diCaffQA (trans).

Figure 3. Exemplary structures of compounds.

Figure 4. Photomicrographs of phloroglucinol-stained sections of stems of M. sinensis,

M. sacchariflorus and M. giganteus.

24Figure 1.

Leaf

12.63

15.15

38.92

36.12

41.5942.30 55.26

44.8753.71

47.6119.34 50.3329.9625.166.15 34.38

22.940.92 9.605.43

14,5

13,14

1115

16,1

7

18

19,2

0

21,2

2 23,2

4

25 2726

8,9

28Abso

rban

ce 3

40 n

m (

AU)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

32.02

39.3626.96

23.2915.02 40.14

35.81

43.7134.70 37.046.08

18.9031.218.75 17.85 45.320.83 48.37 55.001.50

Flower

1 2-4,6 10-1

2 1412

15-1

7

1622

23

8,9

Time (min)

251918

0 10 20 30 40 50 56

Abso

rban

ce 3

40 n

m (

AU)

230000

0

200000

150000

100000

50000

Stem15.02

0.87

35.6210.96

38.62

45.2440.8231.8125.0023.52 42.0227.76 46.28 54.9419.31 47.3622.75 48.18

7.675.42

1

16,1

7

8,9

2

9,11

8 19

22 2327 28

14

2518

7

Abs

orba

nce

340

nm (

AU

)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Leaf

12.63

15.15

38.92

36.12

41.5942.30 55.26

44.8753.71

47.6119.34 50.3329.9625.166.15 34.38

22.940.92 9.605.43

14,5

13,14

1115

16,1

7

18

19,2

0

21,2

2 23,2

4

25 2726

8,9

28Abso

rban

ce 3

40 n

m (

AU)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

32.02

39.3626.96

23.2915.02 40.14

35.81

43.7134.70 37.046.08

18.9031.218.75 17.85 45.320.83 48.37 55.001.50

Flower

1 2-4,6 10-1

2 1412

15-1

7

1622

23

8,9

Time (min)

251918

0 10 20 30 40 50 56

Abso

rban

ce 3

40 n

m (

AU)

230000

0

200000

150000

100000

50000

Stem15.02

0.87

35.6210.96

38.62

45.2440.8231.8125.0023.52 42.0227.76 46.28 54.9419.31 47.3622.75 48.18

7.675.42

1

16,1

7

8,9

2

9,11

8 19

22 2327 28

14

2518

7

Abs

orba

nce

340

nm (

AU

)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Leaf

12.63

15.15

38.92

36.12

41.5942.30 55.26

44.8753.71

47.6119.34 50.3329.9625.166.15 34.38

22.940.92 9.605.43

14,5

13,14

1115

16,1

7

18

19,2

0

21,2

2 23,2

4

25 2726

8,9

28Abso

rban

ce 3

40 n

m (

AU)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Leaf

12.63

15.15

38.92

36.12

41.5942.30 55.26

44.8753.71

47.6119.34 50.3329.9625.166.15 34.38

22.940.92 9.605.43

114,54,5

13,1413,14

11111515

16,1

716

,17

1818

19,2

019

,20

21,2

2 23,2

423

,24

2525 27272626

8,98,9

2828Abso

rban

ce 3

40 n

m (

AU)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Time (min)0 10 20 30 40 50 560 10 20 30 40 50 56

32.02

39.3626.96

23.2915.02 40.14

35.81

43.7134.70 37.046.08

18.9031.218.75 17.85 45.320.83 48.37 55.001.50

Flower

1 2-4,6 10-1

2 1412

15-1

7

1622

23

8,9

Time (min)

251918

0 10 20 30 40 50 56

Abso

rban

ce 3

40 n

m (

AU)

230000

0

200000

150000

100000

50000

32.02

39.3626.96

23.2915.02 40.14

35.81

43.7134.70 37.046.08

18.9031.218.75 17.85 45.320.83 48.37 55.001.50

Flower

11 2-4,62-4,6 10-1

210

-12 1414

1212

15-1

715

-17

16162222

2323

8,98,9

Time (min)

252519191818

0 10 20 30 40 50 56

Abso

rban

ce 3

40 n

m (

AU)

230000

0

200000

150000

100000

50000

0 10 20 30 40 50 560 10 20 30 40 50 56

Abso

rban

ce 3

40 n

m (

AU)

230000

0

200000

150000

100000

50000

Abso

rban

ce 3

40 n

m (

AU)

230000230000

0

200000200000

150000150000

100000100000

5000050000

Stem15.02

0.87

35.6210.96

38.62

45.2440.8231.8125.0023.52 42.0227.76 46.28 54.9419.31 47.3622.75 48.18

7.675.42

1

16,1

7

8,9

2

9,11

8 19

22 2327 28

14

2518

7

Abs

orba

nce

340

nm (

AU

)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Stem15.02

0.87

35.6210.96

38.62

45.2440.8231.8125.0023.52 42.0227.76 46.28 54.9419.31 47.3622.75 48.18

7.675.42

1

16,1

7

8,9

2

9,11

8 19

22 2327 28

14

2518

7

Abs

orba

nce

340

nm (

AU

)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Stem15.02

0.87

35.6210.96

38.62

45.2440.8231.8125.0023.52 42.0227.76 46.28 54.9419.31 47.3622.75 48.18

7.675.42

11

16,1

716

,17

8,98,9

22

9,11

9,11

88 1919

2222 23232727 2828

1414

25251818

77

Abs

orba

nce

340

nm (

AU

)

60000

40000

20000

0

Time (min)0 10 20 30 40 50 56

Time (min)0 10 20 30 40 50 560 10 20 30 40 50 56

25

Figure 2.

Time (min)

Abso

rban

ce 3

40 n

m (

AU)

180000

160000

140000

120000

100000

80000

60000

40000

20000

00 10 20 30 40 50 56

Stem9,64,66

29 30,3

133

3436

-38,

4041

42,1

45-4

749

,50,

5253

,454

-57

598

6010

,61,

62,1

1

12,63

67,1

468 69

,71

72,7

3,18

,74

17,1

574

,75

23,2

5

7622 78 26 27 79 81 82,8

384

Time (min)

Abs

orba

nce

340

nm (

AU)

180000

160000

140000

120000

100000

80000

60000

40000

20000

00 10 20 30 40 50 56

Leaf

88

35,36

39,40

1,43,4446

,47

49,5

1,52

53,5

,455

,56

58

59,8

60,1

0

9,64,65

6

14

70,7

1

72,73

18,7

419

,15

31 32 23,2

5,76

22 77 78

26

27 79 80 85 86

87

26

Figure 3.

Figure 4.

M. sinensis M. sacchariflorus M. x giganteusM. sinensis M. sacchariflorusM. sacchariflorus M. x giganteusM. x giganteus

M. sinensis M. sacchariflorus M. x giganteusM. sinensis M. sacchariflorusM. sacchariflorus M. x giganteusM. x giganteus

R = OH, 5-caffeoylquinic acidR = H, 5-coumaroylquinic acidR = OMe 5-feruloylquinic acid

CO2HRO

OH

HOOH O

HO

R = OH, 5-caffeoylshikimic acidR = H, 5-coumaroylshikimic acid

R = OH, caffeic acid hexosideR = H, coumaric acid hexosideR = OMe ferulic acid hexoside

HOOH

O

OR

OH

CO2H

HOOH

O

CO2HHO

OR

OH

27

Table 1: Quantification of caffeoyl-, p-coumaroyl- and feruloyl- conjugates in the leaves, stems and flowers of M. sinensis and M. sacchariflorus.

Compound No.

Compound M. sinensis M. sacchariflorus Leaf (µg/g

fresh weight) (mol g-1)

Stem (µg/g fresh weight)

(mol g-1)

Flower (µg/g fresh weight)

(mol g-1)

Leaf (µg/g fresh weight)

(mol g-1)

Stem (µg/g fresh weight)

(mol g-1)

Caffeoylquinic acids (CaffQA)a (Mr 354) 7 CaffQA stereoisomer (t) - 62 (0.18) -b - - 1 3-CaffQA (t) 239 (0.68) 62 (0.18) 120 (0.34) 2475 (7.0) 1595 (4.5) 35 3-CaffQA (c) - - - 128 (0.36) 55 (0.16) 14 4-CaffQA (t) 4070 (12) 1053 (3.0) 2039 (5.8) 914 (2.6) - 5 4-CaffQA (c)b 65 (0.18) 17 (0.050) - 55 (0.16) - 9 5-CaffQA (t) 4533 (13) 1173 (3.3) 2271 (6.4) 4002 (11.3) 4002 (11.3) 17 5-CaffQA (c) 788 (2.2) 204 (0.58) 395 (1.1) 518 (1.5) 221 (0.62)

Caffeoylshikimic acids (CaffSA) (Mr 336) 21 3-CaffSA (c) 214 (0.64) - - - 69 (0.21) 26 3-CaffSA (t) 17 (0.051) - - 189 (0.56) - 75 4-CaffSA (t) - - 122 (0.36) 135 (0.40) 20 5-CaffSA (t) 114 (0.34) - - - - 24 5-CaffSA (c) 6.0 (0.018) - - - -

Caffeic acid dimer (Mr 342) 3 Caffeic acid dimer - - 96 (0.28) - -

Caffeoyl-hydroxycitric acid (CaffHydCitA) (Mr 370) 44 2-CaffHydCitA (t) - - - 49 (0.13) 171 (0.46)

Caffeic acid hexosides (CaffA hexoside) (Mr 342) 6 CaffA hexoside (tR 9.9

min) - - 5 (0.015) -

-

28

10 CaffA hexoside (tR 12.8 min)

- - 6 (0.018) 17 (0.05) -

Unknown (Mr 380) 61 caffeoyl (tR 12.1 min) - - - - 527 (1.39) 78 caffeoyl (tR 27.1 min) - - - 98 (0.26) 96 (0.25)

Unknown (Mr 412) 77 Caffeoyl - - - 112 (0.27) -

p-Coumaroylquinic acids (p-CoQA) (Mr 338) 4 3-p-CoQA (t) 11 (0.033) - 11 (0.033) 176 (0.52) 262 (0.78) 19 4-p-CoQA (t) 93 (0.28) 26 (0.077) 92 (0.27) 61 (0.18) - 13 4-p-CoQA (c) 18 (0.053) - - - - 15 5-p-CoQA (t) 298 (0.88) 130 (0.38) 455 (1.35) 217 (0.64) 296 (0.88) 25 5-p-CoQA (c) 234 (0.69 9.0 (0.027) 15 (0.044) 229 (0.68) -

p-Coumaric acid hexosides (p-CoA hexoside) (Mr 326) 2 p-CoA hexoside (t) (tR 7.6

min) - - 406 (1.2)

- - 53 p-CoA hexoside (t) (tR 8.5

min) -

-

- - 74 (0.23)

16 p-CoA hexoside (t) (tR 19.6 min)

- - 770 (2.4) - -

Feruloylquinic acids (FQA) (Mr 368) 11 3-FQA (t) 311 (0.85) 178 (0.48) 91 (0.25) 478 (1.3) 2822 (7.7) 8 3-FQA (c) 110 (0.30) 123 (0.33) 7 (0.02) 117 (0.32) 218 (0.59) 23 4-FQA (t) 328 (0.89) 182 (0.49) 106 (0.29) 78 (0.21) 13 (0.04) 18 4-FQA (c) 44 (0.12) 15 (0.04) 8 (0.02) - 28 (0.08) 22 5-FQA (t) 332 (0.90) 425 (1.15) 75 (0.20) 94 (0.26) 341 (0.93) 27 5-FQA (c) 72 (0.20) 25 (0.07) - - 31 (0.08)

Feruloyl-hydroxycitric acid (FHydCitA) (Mr 384)

29

34 2-FHydCitA (t) (tR 3.9 min)

- - - - 243 (0.63)

Ferulic acid hexosides (FA hexoside) (Mr 356) 12 FA hexoside (t) (tR 13.4

min) - - 21 (0.059) - -

71 FA hexoside (t) (tR 17.1 min)

- - - 43 (0.12) -

Syringic acid hexosides (Hexoside-SA) (Mr 360) 52 SA hexoside (t) (tR 7.9

min) - - -

51 (0.14) 143 (0.40) 56 SA hexoside (t) (tR 9.6

min) -

-

- 22 (0.06) 137 (0.38)

4-Hydroxybenzoic acid hexoside (Mr 300) 30 4-Hydroxybenzoic acid

hexoside (tR 2.9 min) - - - - 200 (0.67)

3,4-Dihydroxybenzoic acid hexoside (Mr 316) 31 3,4-Dihydroxybenzoic

acid hexoside (tR 3.1 min) - - - 196 (0.62) 237 (0.75)

36 3,4-Dihydroxybenzoic acid hexoside (tR 4.7 min)

- - - 319 (1.0) 128 (0.41)

Vanillic acid hexosides (Mr 330) 40 Vanillic acid hexoside

(tR 5.1 min) - - - 60 (0.18) 432 (1.3)

46 Vanillic acid hexoside (tR 6.2 min)

- - - - 284 (0.86)

a CaffQA = caffeoylquininic acid; CaffSA = caffeoylshikimic acid; CaffHydCitA = caffeoyl-hydroxycitric acid; CaffA = caffeic acid; CoQA = coumaroylquinic acid; CoA = coumaric acid; FQA = feruloylquinic acid; FHydCitA = feruloyl-hydroxycitric acid; FA = ferulic acid; SA = syringic acid.

30

c- = not detected in tissue extract / not quantifiable